Nucleic acid-based methods for the detection of microbes in clinical samples can be separated into two broad categories, differing primarily in the lower limit of detection of the target nucleic acid sequence. The first category employs conventional molecular techniques to detect target sequences directly from clinical samples or subcultured microbial isolates. The second category, predicated on nucleic acid amplification technologies, rapidly enriches the target sequences prior to detection thereby bypassing the time, effort and expense of subculturing an isolate prior to analysis. The determination of which approach to employ depends on a number of factors such as cost, labor and the clinical need for rapid results. The nucleic acid molecules of this invention may be advantageously employed in either of the two systems.
Conventional methods for nucleic acid detection rely on physio-chemical methods to foster visualization of the molecules or rely on hybridization methodology employing nucleic acid probes which are labelled with analytically detectable reagents. Examples include: plasmid profiling whereby plasmid DNAs are isolated from microbial isolates and separated by molecular weight by agarose gel electrophoresis; Southern blotting whereby endonuclease-digested DNA is immobilized on supports such as nitrocellulose filters then probed with analytically labeled nucleic acid to detect specific complementary sequences. Analytically detectable reagents for this purposes include radioactive isotopes (e.g., .sup.14 C and .sup.32 P) and non-radioactive reagents such as chemiluminescent materials; DNA dot blots whereby DNA is extracted from a number of microbial isolates by any convenient means and transferred by vacuum filtration to a support and probed as is the case of Southern blotting; and Colony dot blots whereby the colonies are cultured on agar plates, transferred to paper and lysed in situ prior to probing.
Amplification systems rely on the existence of primer nucleic acid molecules of 10-30 nucleotides in length which flank the target region. The primer acts as initiation points for multiple cycles of DNA replication on the region defined by the flanking primers. The Polymerase Chain Reaction employing the Taq DNA polymerase (Mullis and Faloona, Meth. Enzymol. 155:335-350(1987)) is a classic example of an amplification system.
Candida species are well known human pathogens that have been associated with a number of disease states. Most recently, a major concern is hematogenously disseminated infection which is occurring with increased prevalence in postoperative and immunocomprised patients. Identification of such infection can take a minimum of 2 days with an optimal blood culture system, so there is a need for a rapid, sensitive, and specific test to aid in the diagnosis of the disseminated yeast infections. DNA-based diagnostic tests not only are sensitive and specific but also have the potential to decrease the time taken for the laboratory identification of pathogens that are slowly growing or difficult to culture. Early detection and identification of the infecting species in blood or biopsies would facilitate prompt, appropriate treatment.
A number of DNA sequences that are unique to Candida albicans and that may be suitable for diagnostic use have been identified. For example, sequences encoding cytochrome P-450 lanosterol-14.alpha.-demethylase (Buchman, T. G. et al., Surgery 108:338-347 (1990)), mitochondrial DNA (Miyakawa, Y., et al., J. Clin. Microbiol. 30:894-900(1992)), and the secreted aspartyl proteinase (Kanaizuka, I. et al., Jpn. J. Med. Mycol. 34:19-26(1993)) have been used in the PCR-based detection of C. albicans in blood, urine, or cerebrospinal fluid. Nucleic acid probes that hybridize to repeated sequences of C. albicans DNA may provide additional sensitivity, especially when combined with PCR amplification. Recently two DNA fragments were described from the rDNA (genes coding for rRNA) repeat unit of C. albicans for use as DNA probes to detect C. albicans or other yeasts (Holmes, A. R. et al., J. Med. Microbiol. 37:346-351(1992)). One probe hybridized with all fungal DNAs tested, whereas the second was demonstrated to be a C. albicans-specific sequence.
Conserved sequences within repeat regions are attractive targets for PCR-based detection methods because the genome contains multiple copies, thus increasing the proportion of target DNA. If such regions also contain species-specific sequences, the juxtaposition of conserved and unique sequences may enable the complication of fragments to identify both genus and species in a single PCR.
This invention provides a unique set of primers and an EcoRV-NspI probe useful for the detection of a variety of strains of C. albicans and C. stellatoidea.